Structural features of the vitamin K-dependent blood coagulation proteins are being examined by immunochemical methods to gain insight into the special functional features of these proteins. In order to identify the role of individual gamma-carboxyglutamic acid (Gla) residues in prothrombin, partially decarboxylated prothrombin had been prepared by heat decarboxylation of prothrombin. These species are purified and characterized with regard to metal binding properties, Gla content, sequence, and conformation. Conformation specific antibodies specific for prothrombin or for des-gamma-carboxy prothrombin are used to assess tertiary structure. In a separate study, anti-human abnormal prothrombin specific antibodies have been prepared and used to measure abnormal prothrombin in plasma using an RIA. Assay of abnormal prothrombin in plasma obtained from patients with vitamin K deficiency, DIC, liver disease, hemorrhagic disease of the newborn, and those given warfarin have been used to evaluate hepatic carboxylase activity and the adequacy of vitamin K levels in these conditions. Lastly, studies using conformationally-specific antibodies as probes of the structure of prothrombin, abnormal prothrombin, and Factor X will continue. Fragments of these proteins will be prepared proteolytically from the native protein or by solid phase peptide synthesis. These fragments are used to purify antibody subpopulations by sequential immunoabsorption using affinity chromatograpy. These antibody subpopulations are used to determine domains of prothrombin whose structure is metal-stabilized, domains of Factor X that are conformationally-altered during zymogen activation, and regions of abnormal prothrombin which are immunochemically distinct from prothrombin. As a new strategy to the use of antibodies as probes of protein structure, hybridoma cell lines have been raised and their monoclonal antibodies evaluated as reagents for structural studies of the vitamin K-dependent proteins.